Active and passive surveillance for avian influenza virus (AIV) and Newcastle disease virus (NDV) is widespread in commercial poultry worldwide, therefore optimization of sample collection and transport would be valuable to achieve the best sensitivity and specificity possible, and to develop the most accurate and efficient testing programs. A H7N2 low pathogenicity (LP) AIV strain was selected and used as an indicator virus because it is present in lower concentrations in swabbings and thus requires greater sensitivity for detection compared to highly pathogenic (HP) AIV or virulent NDV. Using oro-pharyngeal and cloacal swabs collected from chickens experimentally exposed to the virus, we evaluated the effects of numerous aspects sample collection and transport: 1) swab construction material (flocked nylon, wound/non-flocked nylon, or urethane foam), 2) transport media (brain heart infusion broth [BHI] or phosphate buffered saline [PBS]), 3) media volume (2ml or 3.5ml), 4) transporting the swab wet in the vial or removing the swab prior to transport, or transporting the swab dry with no media, and 5) single swabs versus pooling 5 or 11 swabs per vial. The three most common diagnostic methods for AIV were used with each condition; real-time RT-PCR (rRT-PCR), virus isolation (VI) and commercial antigen detection immunoassays. The effects of pooling 5 or 11 individual swabs on the detection of AIV and NDV by VI and rRT-PCR was also evaluated. Statistically significant differences and consistent trends were observed with some elements; flocked and foam swabs were superior to wound nylon, BHI was better than PBS, and transporting swabs wet was better for virus recovery and detection than transporting them dry. There was no observable difference whether the swab was removed prior to transport or left in the vial. Also, with both AIV and NDV, there was no observed difference in virus detection between pools of 5 and 11 swabs.